Glycolic acid (HOCH2COOH; CAS Registry Number is 79-14-1) is the simplest member of the α-hydroxy acid family of carboxylic acids. Its properties make it ideal for a broad spectrum of consumer and industrial applications, including use in water well rehabilitation, the leather industry, the oil and gas industry, the laundry and textile industry, as a monomer in the preparation of polyglycolic acid (PGA), and as a component in personal care products. Glycolic acid also is a principle ingredient for cleaners in a variety of industries (dairy and food processing equipment cleaners, household and institutional cleaners, industrial cleaners [for transportation equipment, masonry, printed circuit boards, stainless steel boiler and process equipment, cooling tower/heat exchangers], and metals processing [for metal pickling, copper brightening, etching, electroplating, electropolishing]). It has also been reported that polyglycolic acid is useful as a gas barrier material (i.e., exhibits high oxygen barrier characteristics) for packing foods and carbonated drinks (WO 2005/106005 A1). However, traditional chemical synthesis of glycolic acid produces a significant amount of impurities that must be removed prior to use. New technology to commercially produce glycolic acid, especially one that produces glycolic acid in high purity and at low cost, would be eagerly received by industry.
Microbial enzyme catalysts can hydrolyze a nitrile (e.g., glycolonitrile) directly to the corresponding carboxylic acids (e.g., glycolic acid) using a nitrilase (EC 3.5.5.7), where there is no intermediate production of the corresponding amide (Equation 1), or by a combination of nitrile hydratase (EC 4.2.1.84) and amidase (EC 3.5.1.4) enzymes, where a nitrile hydratase (NHase) initially converts a nitrile to an amide, and then the amide is subsequently converted by the amidase to the corresponding carboxylic acid (Equation 2):

Enzymatic hydrolysis of nitriles to glycolic acid for commercial purposes requires production of the enzyme catalyst in high-volume by fermentation. Much of the volume is attributable to the water content of the fermentation broth. Because of said high-volume fermentation broth, storing, and in many cases transporting the fermentation broth comprising the enzyme catalyst, poses both logistical and economic issues. A mechanism for providing ease in storage and transportation of the enzyme catalyst is to isolate the enzyme catalyst from the fermentation broth, immobilize the enzyme catalyst (for example, by entrapment in carrageenan gel), and dehydration of the immobilized enzyme catalyst. The immobilized enzyme catalyst may be rehydrated prior to use for glycolic acid production. However, dehydration and rehydration often result in significant loss in enzyme activity.
The dehydration or drying of immobilized cell catalysts has been previously described. U.S. Pat. No. 5,998,180 describes a process for the production of a dried, immobilized microbial nitrilase, where the Rhodococcus rhodochrous NCIMB 40757 or NCIMB 408333 cells containing said nitrilase retain at least 80% of their initial activity after immobilization in cross-linked polyacrylamide beads, and where the resulting immobilized cell nitrilase retains at least 90% of its initial immobilized activity after the cross-linked polyacrylamide beads are dried to 12% moisture at 60° C. B. DeGiulio et al (World J. Microbial. Biotechnol. 21:739-746, (2005)) describe the immobilization of lactic acid bacteria in calcium alginate, followed by freeze-drying of the resulting immobilized cell catalyst, where at least 72% of the cells retained metabolic activity after freeze-drying. U.S. Pat. No. 5,846,762 describes the dehydration of gelatin beads containing covalently-immobilized cellobiase, and states in column 6, lines 9-11, that calcium alginate and kappa-carrageenan beads, once dehydrated, generally cannot be rehydrated.
None of the methods described immediately above for dehydration or freeze-drying of immobilized enzyme catalysts and subsequent rehydration report an improvement in recovered enzyme activity after rehydration, or improvement in the stability of enzyme activity when the resulting rehydrated enzyme catalyst is employed in a reaction to convert substrate to product, when compared to a comparable rehydrated immobilized enzyme catalyst that was not prepared with glutaraldehyde-pretreated cells.
In addition to loss of enzyme catalyst activity as a result of enzyme catalyst processing, such as in the case of dehydration/rehydration, enzymatic hydrolysis of glycolonitrile to glycolic acid typically requires a substantially pure form of glycolonitrile. Methods to synthesize glycolonitrile by reacting aqueous solutions of formaldehyde and hydrogen cyanide have previously been reported (U.S. Pat. Nos. 2,175,805; 2,890,238; and 5,187,301; Equation 3).

However, these methods typically result in an aqueous glycolonitrile reaction product that requires significant purification (e.g., distillative purification) as many of the impurities and/or byproducts of the reaction (including excess reactive formaldehyde) may interfere with the enzymatic conversion of glycolonitrile to glycolic acid, including suppression of catalyst activity (i.e., decreased specific activity). In particular, it is well known that formaldehyde can create undesirable modifications in proteins by reacting with amino groups from N-terminal amino acid residues and the side chains of arginine, cysteine, histidine, and lysine residues (Metz et al., J. Biol. Chem., 279 (8): 6235-6243 (2004)). Suppression of catalyst activity decreases the overall productivity of the catalyst (i.e., total grams of glycolic acid formed per gram of catalyst), adding a significant cost to the overall process that may make enzymatic production economically non-viable when compared to chemical synthesis. As such, reaction conditions are needed that can help to protect the enzymatic activity against undesirable impurities that decrease the activity of the catalyst.
A method of producing high purity glycolonitrile has been reported by subjecting the formaldehyde to a heat treatment prior to the glycolonitrile synthesis reaction (U.S. Ser. Nos. 11/314,386 and 11/314,905; Equation 3). However, glycolonitrile can reversibly disassociate into formaldehyde and hydrogen cyanide. As such, there remains a need to protect nitrilase activity against the undesirable effects of both formaldehyde and hydrogen cyanide produced by dissociation of glycolonitrile.
U.S. Pat. No. 5,508,181 also describes similar difficulties related to rapid enzyme catalyst inactivation when converting nitrile compounds to α-hydroxy acids. Specifically, U.S. Pat. No. 5,508,181 provides that α-hydroxy nitrile compounds partially disassociate into the corresponding aldehydes, according to the disassociation equilibrium. These aldehydes were reported to inactivate the enzyme within a short period of time by binding to the protein, thus making it difficult to obtain α-hydroxy acid or α-hydroxy amide in a high concentration with high productivity from α-hydroxy nitriles (col. 2, lines 16-29). As a solution to prevent enzyme inactivation due to accumulation of aldehydes, phosphate or hypophosphite ions were added to the reaction mixture. Similarly, U.S. Pat. No. 5,326,702 describes the use of sulfite, disulfite, or dithionite ions to sequester aldehyde and prevent enzyme inactivation, but concludes that the concentration of α-hydroxy acid produced and accumulated even by using such additives is not sufficient for most commercial purposes.
Moreover, U.S. Pat. No. 6,037,155 teaches that low accumulation of α-hydroxy acid product is related to enzyme inactivation within a short time due to the disassociated-aldehyde accumulation. These inventors suggest that enzymatic activity is inhibited in the presence of hydrogen cyanide (Asano at al., Agricultural Biological Chemistry, Vol. 46, pages 1165-1174 (1982)) generated in the partial disassociation of the α-hydroxy nitrile in water together with the corresponding aldehyde or ketone (Mowry, David T., Chemical Reviews, Vol. 42, pages 189-283 (1948)). The inventors address the problem of aldehyde-induced enzyme inactivation by using microorganisms whose enzyme activity could be improved by adding a cyanide substance to the reaction mixture. The addition of a cyanide substance limited the disassociation of α-hydroxy nitrile to aldehyde and hydrogen cyanide. While this tactic provides a benefit to the system, it only addresses one aspect associated with enzyme inactivation in conversion of glycolonitrile to glycolic acid, in that, as stated above, glycolonitrile is known to reversibly disassociate to hydrogen cyanide and formaldehyde, and both are known to negatively effect enzyme catalyst activity.
A separate process has been developed to protect the specific activity of an enzyme catalyst having nitrilase activity when converting glycolonitrile to glycolic acid in the presence of formaldehyde (see copending U.S. application Ser. No. 2009-0111162 incorporated herein by reference), where significant improvements in catalyst activity and stability were achieved by adding an amine protectant to the reaction mixture, or by immobilization of the nitrilase catalyst in or on a matrix that is comprised of an amine protectant, e.g. PEI, polyallylamine, PVOH/polyvinylamine, etc. In that system, the specific activity of the catalyst in the presence of formaldehyde is improved.
Even though many of the above means improved nitrilase catalyst productivity for glycolic acid, a significant decrease in the initial enzymatic activity of the immobilized microbial nitrilase was still generally observed upon use of said catalyst in reactions for the hydrolysis of glycolonitrile, for example, in consecutive batch reactions with catalyst recycle, or in the initial stage of starting up a continuous stirred tank reaction (CSTR) or a fixed-bed column reactor. The problem of significant loss of initial nitrilase activity during hydrolysis of glycolonitrile was addressed in part by pretreating the microbial catalyst with glutaraldehyde prior to immobilization in carrageenan (as described in U.S. Pat. No. 7,695,945 incorporated herein by reference), where a significantly-greater percentage of the initial immobilized microbial nitrilase specific activity (μmoles of glycolonitrile hydrolyzed per minute per gram of catalyst) was retained during the hydrolysis of glycolonitrile to glycolic acid (as the ammonium salt).
U.S. Pat. No. 4,288,552 discloses (column 1, lines 46-49, and column 2, lines 50-55) that glutaraldehyde-sensitive enzymes (such as thiol-enzymes (e.g., nitrilase) and others with an SH group in or very near the active site of the enzyme molecule) are inactivated by thiol-reactive agents such as glutaraldehyde. Therefore, it was not only unpredictable that pretreatment of an enzyme catalyst having nitrilase activity with glutaraldehyde would not result in a significant decrease in microbial nitrilase activity prior to immobilization, but surprisingly, the glutaraldehyde pretreatment was found to benefit enzyme catalyst activity, particularly when the immobilized enzyme catalyst was dehydrated, and subsequently rehydrated prior to use for the hydrolysis of glycolonitrile to glycolic acid. The process of the present invention prevents a significant loss of activity during the dehydration/rehydration steps, and results in a rehydrated immobilized enzyme catalyst with an initial activity and subsequent stability of enzyme catalyst activity during the subsequent used of the rehydrated immobilized enzyme activity for the conversion of glycolonitrile to glycolic acid. This benefit is incorporated into the process described herein which provides for addressing the need for a commercial process, including a dehydration step, for producing an enzyme catalyst having improved specific activity for glycolic acid production upon rehydration.
Therefore, the problem to be solved is the need for a commercially viable process for producing an enzyme catalyst having nitrilase activity for hydrolysis of glycolonitrile to glycolic acid with improved specific activity. More specifically, there is a need for a commercially acceptable process for using an enzyme catalyst having nitrilase activity for the hydrolysis of glycolonitrile to glycolic acid that minimizes loss in enzyme activity resulting from dehydration and rehydration prior to use and resulting from inactivation by impurities or dissociation of reactants.